Investigation of the effect of ethanol concentration on catalase activity

According to the Malaysian Journal of Public Health Medicine 2022, 81% of university students engaged in alcohol consumption and 22.7% maintained unregulated alcohol consumption behaviors. Alcohol stimulates the production of reactive oxygen species (ROS) and denatures catalase. Catalase breaks down ROS in the body to balance the levels of oxidative stress; therefore alcohol consumption contributes to oxidative stress that may result in diabetes, cancer and neuronal damage. (Qi H.M. et al, 2022)

How do different concentrations of ethanol [0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%] affect the catalase activity in potato Solanum Tuberosum measured by the amount of oxygen gas produced after incubating with ethanol for 1 minute and reacting with hydrogen peroxide for 5 minutes?

Catalase is a globular protein and a heme-containing enzyme. A globular protein regulates the rate of reaction by its presence as a catalyst. It contains an active site allowing substrates with a complementary shape identical to the active site to bind to it. An enzyme-substrate complex is produced after the substrate binds to the active site. The enzyme-substrate complex creates an alternative pathway that reduces activation energy for the reaction. In the case of catalase reacting with hydrogen peroxide, hydrogen peroxide molecules bind to catalase active sites, it then interacts with the iron atom from the heme group, which triggers the hydrogen peroxide to be broken down to oxygen and water.

 

Catalase can be found in most living organisms, it acts as an antioxidant enzyme that breaks down cellular hydrogen peroxide. Catalase is found mostly in the liver in the human body. Catalase is important in living cells as it reduces oxidative stress. Oxidative stress is defined as the imbalance of the amount of free radicals and antioxidants in the body. (Dix M, 2018) Catalase reduces oxidative stress by balancing reactive oxygen species [ROS] in cell tissues. Oxidative stress may lead to tissue injury, where an accumulation of ROS may lead to an imbalance of oxidative stress that damages biomacromolecules, including DNA which may result in apoptosis and oncosis. (Authen, R.L. & Davis J.M, 2009) Oxidative stress increases aging, the risk of diabetes, cancer and neuronal damage. This may over-activate the TRPV4 protein channel, leading to heightened rates of renal dysfunction and tissue damage. (Hong. Z. et al, 2016) Hydrogen peroxide is a cellular ROS produced naturally in the body as a byproduct of cellular respiration. Catalase breaks down hydrogen peroxide (2H₂O₂ → 2H₂O+O₂) into oxygen and water reducing the accumulation of hydrogen peroxide in cells. Hydrogen peroxide is a ROS that may cause oxidative damage towards cellular components. (Ransy. C. et al, 2020) Therefore, catalase is crucial in the role of reducing oxidative stress in the body.

 

Ethanol is the most common form of alcohol found in alcoholic beverages. Different alcohols have different concentrations of ethanol, usually, people consume ranges from 1% to 40% in the form of beer, wine, liquor and spirits. (Yerby. N, 2023).The breakdown of alcohol after consumption occurs in the liver, where catalase levels are the highest. Catalase is located in peroxisomes that undergo oxidative reactions using the molecular oxygen obtained after breaking down cellular hydrogen peroxide. Ethanol is metabolized by catalase to form acetaldehyde, then acetaldehyde turns into acetate by mechanisms of aldehyde dehydrogenase; ethanol metabolism also creates ROS as byproducts. Therefore ethanol in the process of catalase breaking down hydrogen peroxide becomes a competitive inhibitor by occupying catalase active sites. (Biomolecule-Enzymes, n.d.) This prevents the hydrogen peroxide molecule from binding to catalytic sites and therefore can’t be broken down. Chronic alcohol consumption may lead to sustained levels of catalase inhibition, and increased levels of ROS contributing to oxidative stress may cause feedback inhibition where the synthesis of signaling pathways can cause the inhibition or suppressed expression of the enzyme catalase. (Choi et al., 2009) The observation of ethanol inhibition may be shown through the volume of oxygen gas produced from the breakdown of hydrogen peroxide

H_o - As the concentration of ethanol [0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%] increases, it will show no significant difference in the volume of oxygen gas produced from catalase enzymes breaking down hydrogen peroxide. Therefore, no impact of ethanol concentration on catalytic activity would be observed.

 

H₁ - As the concentration of ethanol [0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%] increases, the volume of oxygen gas produced would decrease after 5 minutes. This occurs due to more ethanol molecules present to inhibit catalase active sites, thereby decreasing the rate of breaking down hydrogen peroxide.

• 8x Potato Solanum tuberosum
• 1x blender
• 9x airtight bottles
• 1x refrigerator
• 2x 25 cm³ measuring cylinder (± 0. 25ml)
• 2x 10 cm³ measuring cylinder (± 0. 1ml )
• 2x test tube bung
• 1x test tube bung with hole
• Pipettes
• 1x tiny spoon
• 1x water bath
• 1x large container
• 1x clamp stand
• 1x scotch tape
• 1x permanent marker
• 1x timer
• 500ml 95% ethanol
• 500ml 3% Hydrogen Peroxide

Preparing puree solution [100%]

• Cut up a potato into smaller pieces, then blend up in a blender. The mixture should be watery with potato flesh residue
• Transfer the potato mixture into a bottle with an airtight lid, do not mix the mixture vigorously in this process
• Put the bottle into the refrigerator from 2°C to 8°C to preserve and immobilize the catalase.

• Add water to the container and leave a small gap
• Get a 25cm³ measuring cylinder and use masking tape to cover up half of the cylinder from 0cm³ to 15cm³ - ensure the tape does not cover the numbers and leaves a gap for the student to observe
• Fill a 25cm³ measuring cylinder with water
• Set the measuring cylinder filled with water, turn it upside down and put it into the container. Ensure that there is no air bubble in the measuring cylinder
• Use a clamp stand to secure the measuring cylinder in position
• Connect a rubber tube to a test tube bung
• Insert the other end of the rubber tube into the measuring cylinder; ensure that the tube end is aimed inside the measuring cylinder. Continue to ensure that there are no air bubbles in the measuring cylinder

• Ensure a dry environment around the water bath
• Switch on the power supply connected to the water bath
• Fill the water bath with distilled water above the heating element
• Switch on the water bath and set the thermostat to 37°C to imitate the conditions in the human body and allow catalase to reach an optimum temperature to be active
• Fix a thermometer in the water bath to ensure the temperature of the water bath is constant with the desired temperature
• Wait until the water bath reaches the desired temperature
• Add a conical flask into the water bath

• Use the formula [original ethanol concentration × x = volume of solution produced × percentage of ethanol wanted] to calculate the amount of ethanol needed to produce the desired concentration
• Calculate for x, then pour out the according measurement of 95% alcohol in an airtight bottle
• Use the volume of the solution produced and minus the x, which would be the amount of water needed to dilute ethanol
• Measure the amount of water calculate and pour into the airtight bottle
• Immediately tighten the lid and lightly swirl the ethanol solution
• Store the ethanol solution in the refrigerator [3°C~5°C] to reduce oxidation

• Prepare the concentrations of the ethanol solution of 5%, 10%, 15%, 20%, 25%, 30%, 35% and 40%, respectively pour each into an airtight bottle to avoid oxidation and evaporation
• Pour 20 cm³ of the prepared potato puree solution into the conical flask set in the water bath
• Seal the conical flask with a bung to prevent oxidation of the potato puree, and leave it for 2 minutes for catalase to reach optimal temperature with maximum enzymatic activity
• Add 5ml of 5% ethanol solution into the conical flasks, lightly swirl the mixture and leave for 1 minute for the ethanol solution to mix and be absorbed by the potato puree
• Pour 4ml of 3% hydrogen peroxide into the conical flask, and immediately connect the conical flask with the bung connected to the rest of the apparatus
• Lightly swirl the conical flask to mix the hydrogen peroxide with the rest of the potato solution mixture
• Start the stopwatch
• At respective time intervals [15, 30, 45, 60 at each minute]
• and observe the amount of oxygen gas produced in the measuring cylinder
• Record the volume of gas produced in the measuring cylinder at 15-second intervals for 5 minutes
• Repeat steps 3 to 8 for each ethanol concentration

• Wear safety goggles, a lab coat and closed toe shoes to avoid direct contact with ethanol and hydrogen peroxide
• Tie up hair in the laboratory to avoid contact with chemicals

• Work with ethanol and hydrogen peroxide in a well-ventilated area, avoid inhalation as both substances cause harm towards the central nervous system and the respiratory system after ingestion
• Ethanol is highly flammable. Use ethanol in a well-ventilated area, far from any heat sources
• Skin and eye irritation: Ethanol can be irritating to the skin and eyes. Avoid direct contact with ethanol; wear gloves and safety goggles when handling it.
• Ethanol and hydrogen peroxide can create a hazardous situation if spilt. Clean up spills immediately, and dispose of the waste accordingly.

• After 5 minutes of reaction, the student noticed an accumulation of small bubbles above the potato puree and hydrogen peroxide mixture indicating the production of gas from the reaction
• After 5 minutes of reaction, the colour of the potato puree solution turned into a slightly darker hue compared to before the reaction, indicating the oxidation of the potato catalase enzyme (refer to Appendix B)

• Refer to Appendix A for the 4 trials of the collection of the Volume of Oxygen Gas Produced in 15 Second Intervals for 5 Minutes (cm³)

Refer to Appendix C for outlier calculations. To calculate outliers for the final volume of oxygen gas produced at different ethanol concentrations at 5 minutes, the formula below has been applied -

 

Q1 = first lower quartile

 

Q3 = third upper quartile

 

IQR = Q3 - Q1

 

Lower Fence = Q1 - (1.5 × IQR)

 

Upper Fence = Q3 + (1.5 × IQR)

 

The calculations from Appendix C indicate that there are no outliers for each ethanol concentration of all 4 trials. Therefore, the processed data would include all data sets from each trial.

The reaction rate percentage of the catalase breaking down hydrogen peroxide can be calculated with the formula below. The calculations done would use the values of the average volume of gas produced from figure 10. Refer to Appendix D for the values of the calculations done.

 

\(\text{Rate of Change in Percentage} =[\frac{final \ value \ - initial\ value}{Time}×100\%]\)

The scatter-plot above shows a decreasing trend of the rate of reaction of oxygen gas production at each ethanol concentration. As ethanol concentration increases, the rate of reaction between catalase and hydrogen peroxide decreases, this is seen in the resulting volume of oxygen gas produced.

The calculation of the Pearson correlation coefficient will show the strength of the correlation between the 15-second time intervals and the volume of oxygen gas produced at different ethanol concentrations. It will show between the range of -1 to 1; all calculations will be done with the TI-84 Plus CE calculator.

 

Figure 11 - Pearson Correlation Formula ((Patil, 2023))

 

\(r=\frac{\sum(x_i-\overline{x}) (y_i-\overline{y})}{\sqrt{\sum(x_i-\overline{x})^2}\sum(y_i-\overline{y})^2}\)

 

Where,

 

r = Pearson Correlation Coefficient

 

\(x_i\) _{= × variable samples}

 

_{\(\overline{x}\) = mean of values in x variable}

 

\(y_i\) _{= y variable sample}

 

_{\(\overline{y}\) = mean of values in y variable}

As seen in Figure 6, the average rate of reaction of oxygen gas production decreases as the ethanol concentration increases, indicating a strong negative correlation between the two variables. As predicted in the hypothesis, the increased concentration of ethanol will result in a decrease in oxygen gas production; it demonstrates a negative gradient with a linear relationship suggesting that the two variables are inversely proportional. The strong relationship between the data is supported by the Pearson correlation coefficient calculated. The correlation coefficient of the relationship between ethanol concentration and volume of oxygen gas produced was almost perfect with a r-value of -0.990; which is close to -1 suggesting a strong negative correlation between both variables.

 

In Figure 8, a steady increase in oxygen gas production of different ethanol concentrations can be observed. The highest volume of production was when ethanol concentration was at 0%, and oppositely the lowest value of gas production at 5.9cm³ was seen at ethanol concentration at 40% . Other ethanol  concentrations such as 5% showed a slower rate of oxygen production compared to 10% and 15% at times 60 to 240 seconds, the final volume of gas indicated by 5% was lower than 0% but higher than 10% and 15%. This again indicates as catalase is exposed to increasing ethanol concentrations, the volume of oxygen gas produced by catalase breaking down hydrogen peroxide is decreased.

From the data displayed in the experiment, it can be concluded that as ethanol concentration increases, the production of oxygen gas decreases, indicating a lower rate of catalase activity with a lower rate of hydrogen peroxide breaking down. This suggests that the null hypothesis is rejected and the alternative hypothesis is accepted.

 

The data and statistical test produced both support and portray a strong correlation between the two variables. This occurs due to the higher concentration of ethanol containing more ethanol molecules in the same volume, the more ethanol molecules, the higher the chance where ethanol molecules bind to the catalase active sites. This decreases the availability of active sites for hydrogen peroxide to bind to therefore resulting in a decrease in catalytic activity. The decrease of hydrogen peroxide binding to catalase active sites to form an enzyme-substrate complex, the volume of oxygen gas produced is also reduced as hydrogen peroxide was not fully broken down. A similar result was seen in the results of a research. The study showed the inactivation of the cellulase by ethanol; enzyme loss was minimal around 0-40 g/L while enzyme inactivity increases as the concentration increases to 80 g/L with a loss of 75% enzymatic activity. (L.R, 2011) Another research paper has observed that catalase activity increases in low concentrations of ethanol but decreases in higher concentrations of ethanol. (Das & Vasudevan, 2005) Both research papers show a similar trend observed from the study the student has conducted, that as ethanol concentration increases, enzyme activity decreases observed from the volume of oxygen produced.

An extension for this experiment is to test the effects of different types of alcohol such as methanol, propanol, butanol and phenyl ethanol that are present in beer, spirits and wine. Instead of testing the effect of different ethanol concentrations, the student can test different types of alcohol with increasing concentrations to mimic the more realistic situation in terms of a person who consumes alcoholic beverages and the effect of enzymatic activity in the body. The experimental procedure would differ from the procedure noted in this study. Therefore the research question would be “How do different types of alcohol at different concentrations affect the rate of alcohol dehydrogenase breakdown” The enzyme used can be switched to alcohol dehydrogenase, which breaks down alcohol into acetaldehyde. Then the presence and concentration of acetaldehyde can be tested through high-performance liquid chromatography. The effect of different alcohol types on alcohol dehydrogenase breakdown can be measured by the rate of reaction of each alcohol type and compared. This different procedure may give more insights and evidence of alcohol's effect on the rate of reaction of enzymatic activity; leading to more insight on the effect of alcohol on oxidative stress in the body and information regarding the dangers of overconsuming alcoholic beverages.

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4 Trials of The Volume of Oxygen Gas Produced in 15 Second Intervals for 5 Minutes (cm³)

Photos of the Potato Puree Solution Colour Change Before and After the Reaction

Calculation Using T1-84 Plus CE of Outliers for The Amount of Oxygen Gas Produced (cm³) After 5 Minutes at All Ethanol Concentrations

Average Rate of Reaction Change In Percentage and Max-Min Bar Calculation